ABSTRACT Cerebral small vessel disease (SVD) affects over half of Americans over 65 and is a major risk factor for stroke and dementia. Although the pathological changes in blood vessels in SVD have been described for decades, the precise molecular defects are unclear. CADASIL is the most common inherited form of SVD and is caused by mutations in NOTCH3. We investigate the pathogenesis of CADASIL as a potential window into uncovering targetable mechanisms of vascular degeneration of the brain. Molecular mapping of affected families has provided important clues to the molecular pathways leading to arterial degeneration in CADASIL. The SVD-causing mutations in NOTCH3 either create or delete one cysteine residue, supporting the hypothesis that disulfide dependent conformational alterations in NOTCH3 trigger pathology of brain small vessels. In new preliminary data, we use a set of new NOTCH3 antibodies, including monoclonal probes, that bind to conformations of NOTCH3 that are strongly expressed in pathologically affected CADASIL arteries. These antibodies specifically bind to reduced NOTCH3 protein; other denaturants fail to induce the CADASIL conformational change. Site-directed mutagenesis of NOTCH3 demonstrates that the CADASIL conformation of NOTCH3 is generated after mutation of multiple cysteines, leading us to hypothesize that the unique NOTCH3 protein expressed in CADASIL results from reduction of more than one disulfide bond. We call this protein Multiple Reduced Cysteine NOTCH3 (mrc-N3). The experiments of this proposal aim to characterize mrc-N3. We propose to test the following hypotheses (Figure 1): (1) mrc-N3 is generated from reduction of at least two disulfide bonds and genesis of mrc-N3 is facilitate by NOTCH3 binding proteins from the vessel wall; (2) mrc-N3 activates transcription of disease related proteins and triggers smooth muscle cell stress pathways; (3) mrc-N3 functions in vivo to cause vascular dysfunction and pathology. These Aims will be tested using biochemical techniques incorporating purified NOTCH3 proteins, cell culture using primary human cerebral smooth muscle, pathological analysis of unique tissue resources available to our lab, and in vivo testing of mrc-N3 function in genetically modified mice. Successful execution of these studies will shed new light on key molecular mechanisms of small vessel disease.